Shi Xun-Long, Feng Mei-Qing, Shi Jian, Shi Zhi-Hui, Zhong Jiang, Zhou Pei
Department of Drug Biosynthesis, School of Pharmacy, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China.
Protein Expr Purif. 2007 Jul;54(1):24-9. doi: 10.1016/j.pep.2007.02.008. Epub 2007 Feb 21.
Catalase is one of the antioxidant enzymes and is involved in many pathophysiologic processes and human diseases. This study focused on high-level expression and purification of recombinant catalase in Pichia pastoris. The cDNA encoding catalase was cloned by RT-PCR from Fetal liver of Homo sapiens. After PCR and construction of expression vector pPIC9K-CAT, human catalase was expressed highly in P. pastoris yeast SMD1168 and secreted into the culture medium. The secreted catalase was purified to a purity of 95% by ammonium sulfate fractionation, anionic exchange-chromatography, and Macro-prep Ceramic Hydroxyapatite with a overall yield of 60%. This study provides a new method for large-scale expression and purification of recombinant protein catalase.
过氧化氢酶是抗氧化酶之一，参与许多病理生理过程和人类疾病。本研究聚焦于在毕赤酵母中高效表达和纯化重组过氧化氢酶。通过RT-PCR从智人胎儿肝脏中克隆出编码过氧化氢酶的cDNA。经PCR及构建表达载体pPIC9K-CAT后，人过氧化氢酶在毕赤酵母SMD1168中得到高效表达并分泌到培养基中。分泌的过氧化氢酶经硫酸铵分级沉淀、阴离子交换色谱和大孔陶瓷羟基磷灰石纯化，纯度达到95%，总产率为60%。本研究为重组蛋白过氧化氢酶的大规模表达和纯化提供了一种新方法。
